1. Volume 157, Issue 3, Pages 580-594 (April 2014)
Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-like Cells 
Mario L. Suvà, Esther Rheinbay, Shawn M. Gillespie, Anoop P. Patel, Hiroaki Wakimoto, Samuel D. Rabkin, Nicolo Riggi, Andrew S. Chi, Daniel P. Cahill, Brian V. Nahed, William T. Curry, Robert L. Martuza, Miguel N. Rivera, Nikki Rossetti, Simon Kasif, Samantha Beik, Sabah Kadri, Itay Tirosh, Ivo Wortman, Alex K. Shalek, Orit Rozenblatt-Rosen, Aviv Regev, David N. Louis, Bradley E. Bernstein 
Cell 
Volume 157, Issue 3, Pages (April 2014)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Epigenetic Landscapes Distinguish Functionally Distinct GBM Models
(A) Survival curves for xenotransplanted mice. GBM cells (MGG8) grown as gliomaspheres in serum-free conditions propagate tumor in vivo, whereas serum-differentiated cells fail to do so.
(B) Flow cytometry of MGG8 TPCs shows positivity for the GBM stem-like markers SSEA-1 and CD133, whereas serum-differentiated cells do not.
(C) Cells grow in serum as adherent monolayers and express the differentiation markers GFAP (astroglial), β III tubulin (neuronal), MAP-2 (neuronal), and GALC (oligodendroglial).
(D) Xenografted tumors from MGG8 TPCs (left) are invasive, crossing the corpus callosum (boxed region) and infiltrating along white matter tracks (arrowhead). At high magnification, the cells are atypical, and mitotic figures are evident (arrow). Xenografted tumors from MGG4 TPCs (right) are more circumscribed but also infiltrate adjacent parenchyma (boxed region, arrowhead). At high-magnification areas of necrosis (∗) and mitotic figures (arrow) are readily identified. LV, lateral ventricle.
(E) Heatmap depicts genomic intervals (rows) enriched for H3K27ac in tumor models (cyan: high signal), clustered into groups of TPC-specific, DGC-specific, and shared regulatory elements. Shared elements tend to be located proximal to promoters, whereas the vast majority of TPC- and DGC-specific elements are distal. Motif analyses predict binding sites for TF families within each set of elements.
See also Figure S1.
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4. Figure 2
Candidate Regulators for the Specification of Alternate Epigenetic States in GBM
(A) A set of 19 TPC-specific TFs is identified based on RNA-seq expression (red, high; blue, low) and promoter H3K27ac signals (green, high) in TPCs and DGCs (TSS, transcriptional start site). TF family is indicated at right.
(B) Western blots confirm exclusive protein expression in TPCs for selected TFs. Bottom indicates tubulin loading control.
(C and D) (C) ChIP-seq tracks show H3K27ac signals for loci encoding TPC-specific TFs OLIG1, OLIG2, and SOX2 or (D) the differentiation factor BMP4 in the respective GBM models. TPC-specific TF loci are enriched for TPC-specific regulatory elements.
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5. Figure 3 A Core TF Network for Tumor-Propagating GBM Cells
(A) Data points indicate percentage of single-cell DGCs capable of forming spheres in serum-free conditions. Each of the 19 TFs in Figure 2A was tested alone (first column, “single TF”) in combination with POU3F2 (second column) or in combination with POU3F2 and SOX2 (third column). HLH family TFs were also tested in combination with POU3F2, SOX2, and SALL2 (fourth column) based on an enrichment of HLH motifs in regulatory elements that failed to activate in 3TF-induced DGCs. TF combinations that enhanced in vitro spherogenicity (blue) were selected for in vivo testing. Error bars represent SEM in duplicate experiments.
(B) Flow cytometry profiles show expression of the stem cell marker CD133 for DGCs induced by the single, double, triple, and quadruple TF combinations with the highest in vitro sphere-forming potential.
(C) For TF combinations with in vitro spherogenic potential (blue in A), 100,000 cells were injected in the brain parenchyma (n = 4 mice per TF combination). Survival curve is shown for this in vivo tumor-propagation assay. Only the quadruple TF combination POU3F2+SOX2+SALL2+OLIG2 initiated tumors in mice.
(D) Tumor histopathology shows characteristic features of glioblastoma, including necrotic areas (∗) and crossing of corpus callosum (boxed area). At high magnification, cells show atypical features, and mitotic figures are evident (arrows). LV, lateral ventricle.
(E) Secondary TPC sphere cultures (iTPC) derived from xenotransplant tumors express the stem-cell marker CD133.
(F) Contrast field image of iTPC spheres.
(G) Left, bar graph shows iTPC and TPC proliferation rates measure by BrdU incorporation. Right, data points indicate percentage of single cells capable of serial sphere formation in three consecutive passages in serum-free conditions. Self-renewal properties and proliferation of iTPCs are comparable to corresponding TPCs. Error bars indicate SEM based on two data points.
(H) Orthotopic serial xenotransplantation in limiting dilution shows that as few as 50 MGG8 iTPC are sufficient to initiate tumors.
(I) Data points indicate in vitro sphere formation of MGG4 TPCs infected with lentivirus containing shRNA for POU3F2, OLIG2, or SALL2, as compared to control (two hairpins per TF). Error bars represent SEM based on two data points.
(J) Survival curve depicts in vivo tumor-propagating potential of MGG4 TPCs infected with POU3F2 shRNA, SALL2 shRNA, OLIG2 shRNA, or control shRNA.
See also Figures S2, S3, and S4.
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6. Figure 4 Core TFs Reprogram the Epigenetic Landscape of DGCs
(A) Left, heatmap depicts H3K27ac signals for TPC-specific, DGC-specific, or shared regulatory elements defined in Figure 1E. Relative to control vector infected DGCs, iTPCs gain H3K27ac over TPC-specific elements and lose H3K27ac over DGC-specific elements, which is consistent with genome-wide reprogramming of the epigenetic landscape. Right, pie charts show fraction of regulatory elements (dark cyan) in each set with H3K27ac in iTPC.
(B) RNA-seq expression and promoter H3K27ac levels at promoter are shown for TPC-specific TFs defined in Figure 2A (NES, Nestin).
(C) Hierarchical clustering of MGG8 DGCs, TPCs, and replicate iTPCs (iTPC1/2) by H3K27ac ChIP-seq signal.
(D) RNA-seq (3′ end) tracks show that core TF mRNAs in iTPCs include 3′ UTRs (shaded in gray). This indicates that endogenous loci are reactivated in iTPCs as the exogenous vectors lack 3′ UTRs.
(E) H3K27ac signal tracks for loci encoding core TFs show that endogenous regulatory elements (highlighted with gray shading) are reactivated in iTPCs.
(F and G) (F) Serum-induced differentiation leads iTPCs to convert to an adherent phenotype, to upregulate differentiation markers GFAP, β III tubulin, MAP-2, and GALC and (G) to lose CD133 expression.
(H) Western blots confirm serum-induced differentiation of iTPCs leads to downregulation of core TFs. Bottom, tubulin loading control. These data indicate that the core TFs can reprogram DGCs into stem-like GBM cells, which have an epigenetic landscape similar to TPCs that is sustained by endogenous regulatory programs.
See also Figure S2.
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7. Figure 5
All Four Core TFs Are Coordinately Expressed in a Subset of Primary GBM Cells with Stem-like Markers
(A) Quadruple immunofluorescence for core TFs in three human GBM samples shows coexpression in a subset of cells; shown at right are the fractions of SOX2+ cells that express each other individual TF or all four TFs in each tumor. Error bars represent SD of positive cells across 10 fields examined.
(B) Flow cytometry analysis from acutely resected GBM tumors. A majority of cells positive for the four core TFs express the stem-cell marker CD133. Enrichment is significantly greater than for SOX2-expressing cells.
(C) Heatmap shows H3K27ac signal from three freshly resected GBM tumors for regulatory elements defined in Figure 1E. Right, pie charts show fraction of regulatory elements (dark cyan) in each set with H3K27ac. TPC-specific elements show significant enrichment, which is consistent with a TPC-like regulatory program in a subset of cells.
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8. Figure 6 TF Network Reconstruction and Targeting
(A) ChIP-seq signal for core TFs in TPCs (MGG8) is shown for regulatory element intervals defined in Figure 1E. Preferential binding is evident at TPC-specific regulatory elements.
(B) Pie charts indicate proportion of TF binding sites that coincide with the indicated sets of regulatory elements.
(C) Sequence motifs identified in TF ChIP-seq peaks. With the exception of SALL2 (see Results and Figure S5), motifs correspond to the expected class of TFs, further validating ChIP-seq experiments.
(D) Model for core TF regulatory interactions reconstructed from binding profiles and expression data (see Results and Experimental Procedures). Other TFs defined in Figure 2A (green) and chromatin regulators (red) are highlighted.
(E) Signal tracks depict core TF binding over TPC-specific regulatory elements within loci containing the corresponding TF genes.
See also Figure S5.
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9. Figure 7 The LSD1-RCOR2 Chromatin Complex Is Essential for GBM TPCs
(A) Plots depict LSD1 and RCOR2 RNA-seq expression values for TPCs and DGCs. Error bars indicate SEM based on three data points.
(B) Western blot for RCOR2 (MGG8 TPC and DGC lysates) confirms exclusive expression in TPC.
(C) Western blot for LSD1 on RCOR2 immunoprecipitate indicates coassociation between the two proteins in TPCs.
(D) Signal tracks depict TF binding and H3K27ac enrichment in the RCOR2 locus. OLIG2 binds a TPC-specific regulatory element in the locus.
(E) Survival curve of mice injected with DGCs induced with the combination of POU3F2+SOX2+SALL2+RCOR2 indicates that RCOR2 can substitute for OLIG2 in the cocktail.
(F) Coronal section of a xenografted GBM tumor (dashed line) established from iTPCs reprogrammed with the POU3F2+SOX2+SALL2+RCOR2 combination.
(G) Representative images of TPCs and DGCs infected with LSD1 shRNA show reduced viability specifically in the TPCs.
(H) Bar graphs depict percent viability for MGG4 TPCs or DGCs infected with control shRNA or two different LSD1 shRNAs. LSD1 depletion causes decreased viability in TPCs but has no effect on DGCs. Error bars represent SEM in duplicate experiments.
(I) Data points indicate in vitro sphere formation of MGG4 TPCs infected with lentivirus shRNA for LSD1 (two hairpins), compared to control in three serial passages. Error bars indicate SEM based on two data points.
(J) Graph depicts percent viability for TPCs and DGCs (MGG4 and MGG8) and primary astrocytes (NHA) exposed to increasing doses of the synthetic LSD1 inhibitor S2101. A representative image of TPCs exposed to 20 μM S2101 for 96 hr is shown below. Error bars indicate SEM in duplicate experiments.
(K) Survival curve depicts in vivo tumor-propagating potential of MGG4 TPCs infected with LSD1 shRNA (two hairpins) or control shRNA. These data suggest that the RCOR2/LSD1 complex is essential for stem-like TPCs and thus represents a candidate therapeutic target for eliminating this aggressive GBM subpopulation.
See also Figure S4.
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10. Figure S1
Additional Characterization of Our Models, Related to Figure 1
(A) GBM cells (MGG4) grown as gliomaspheres in serum-free conditions propagate tumor in vivo while serum-differentiated cells fail to do so.
(B) Serum-grown cells grow as adherent monolayers and express the differentiation markers GFAP (astroglial), beta III tubulin (neuronal), MAP-2 (neuronal) and GalC (oligodendroglial).
(C) Hierarchical clustering of H3K27ac ChIP-Seq signal separates GBM TPCs from DGCs (see methods).
(D) Distance of marker gene signature in TPCs to TCGA-defined centroids for each molecular subtype (Verhaak et al., 2010). Lower distance indicates greater similarity to respective subtype.
(E) Expression of PTEN by RNA-seq in our three matched lines of TPCs and DGCs shows expression levels comparable or higher to primary human astrocytes (NHA). Error bars indicate SEM based on three data points.
(F) Western blot for PTEN shows expression of the protein in MGG4 TPCs and MGG8 TPCs (Chen et al., 2010).
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11. Figure S2
Reprogramming of the H3K27ac Epigenomic Landscape, Related to Figures 3 and 4
(A) Diagram shows percentage of H3K27ac peaks in the three sets of regulatory elements as defined in Figure 1E at different stages of reprogramming, showing a decrease of DGC specific and an increase of TPC-specific elements during reprogramming.
(B) Percentage of TPC-specific regulatory elements (relative to shared elements) that gain H3K27ac after single TF induction in DGCs. Only SOX2 and POU3F2 are capable of activating TPC-specific elements independently.
(C) Hierarchical clustering of H3K27ac ChIP-Seq tracks in MGG8 TPCs, DGCs and at different stages of reprogramming. iTPCs cluster with TPCs.
(D) De novo motif analysis of H3K27ac sites present in TPCs but not partially reprogrammed cells (POU3F2, SOX2, SALL2).shows enrichment of an HLH-class motif.
(E) Representative images of H3K27ac ChIP-Seq tracks at stages of reprogramming. SOX2 and POU3F2 are displayed as representative examples of loci that get activated during reprogramming.
Cell  , DOI: ( /j.cell )
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12. Figure S3
BMP4 Differentiation Downregulates Core TFs and Can Be Reversed by TF Induction, Related to Figure 3
(A) qRT-PCR measurements of mRNA for POU3F2, SOX2, OLIG2 and SALL2 in MGG8 TPCs, TPCs differentiated in serum for 72 hr (FCS 72h) and differentiated with BMP4 for 72 hr (BMP4 72h). Error bars indicate SEM based on three data points.
(B) Left: flow cytometry for CD133/isotype control in MGG8 TPC control or treated with BMP4. Right: flow cytometry for CD133/isotype control of BMP4-differentiated MGG8 TPCs infected with inducible lentiviruses encoding POU3F2, SOX2, OLIG2 and SALL2. Induction by doxycycline results in higher CD133 expression.
(C) Left: BMP4-differentiated MGG8 TPCs rapidly adhere and differentiate, as previously reported. Middle and Right: BMP4-differentiated MGG8 TPCs infected with inducible lentiviruses encoding POU3F2, SOX2, OLIG2 and SALL2 cultured in the absence or presence of doxycycline. Induction of TF expression generates spheres in vitro. Collectively, these data support a general role for these TFs in the stemness of GBM cells responding to different differentiation stimuli.
Cell  , DOI: ( /j.cell )
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13. Figure S4
qRT-PCR Measurements of shRNA Knockdowns, Related to Figures 3 and 7
(A) qRT-PCR measurements of mRNA for POU3F2, OLIG2 and SALL2 in MGG4 TPCs infected with control lentivirus shRNA or with hairpins specifically targeting the corresponding mRNA, showing downregulation of each TF with two different hairpins. Error bars indicate SEM based on three data points.
(B) qRT-PCR measurements of mRNA for LSD1 in MGG4 TPCs and DGCs infected with control lentivirus shRNA or with hairpins specifically targeting LSD1, showing similar downregulation in TPCs and DGCs with two different hairpins.
Cell  , DOI: ( /j.cell )
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14. Figure S5
Western Blots, Immunoprecipitation, and Coimmunoprecipitation Assays of SOX2, POU3F2, SALL2, and OLIG2 in GBM TPC, Related to Figure 6
(A) Western blot and immunoprecipitation experiments using MGG8 TPC lysates show specificity of the antibodies for their corresponding TF. Arrow points to corresponding TF band.
(B) Venn diagram depicts numbers of TF peaks at regulatory elements and overlap among these sites.
(C) Western blot for SALL2 on MGG8 TPC lysate and after immunoprecipitation (control IgG, SOX2 IP, SALL2 IP, POU3F2 IP and OLIG2 IP) highlights interaction between SALL2 and SOX2 in GBM TPCs.
Cell  , DOI: ( /j.cell )
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